1. Field of the Invention
The invention relates to modulation of gene expression. In particular, the invention relates to modulation of gene expression through an antisense approach.
2. Summary of the Related Art
Regulation of gene expression is a complex process, and many aspects of this process remain to be understood. Aberrant gene expression appears to be responsible for a wide variety of inherited genetic disorders, and has also been implicated in numerous disease states including pathological conditions stemming from tumorigenic growth. A great deal of cancer related research pertains to the elucidation of the roles and interaction of tumor suppressor genes and oncogenes.
Several tumor suppressors have been identified. Marshall et al., Cell 64:313-326 (1991) teach that the WT1 gene was among the first tumor suppressors to be identified and isolated. Coopers et al., Cancer Invest. 12(1):57-65 (1994) disclose that the WT1 gene product is a protein with four zinc fingers suspected to be a transcription factor. Anderson and Spandidos Onco-Suppresso (1990) disclose the NF1 gene, another tumor suppressor, involved in the development of neurofibromatosis functioning as a GTPase-activating protein for the GTP-binding protein p21.sup.ras. In addition, Sager et al., Science 246:1406-1412 (1989) disclose several genes involved in the development of colon cancer, namely DCC, MCC and APC (FAP) suggesting that their products might also perform tumor suppressor functions.
To date however, the best characterized tumor suppressors are the RB and the p53 gene products.
Levine, Bioessays 12(2):60-66 (1990) teaches RB gene inactivation in retinoblastoma. Notably, Levine et al., Nature 351:453-456 (1991), Weinberg et al., Neur. 11:191-196 (1991), and Williams et al., Nature Genet. 7:480-484 (1994), teach RB gene inactivation in many other tumor types including breast tumors, bladder carcinoma, and lung tumors.
Levine et al., Nature 351:453-456 (1991) have disclosed that the p53 tumor suppressor gene encodes a phosphoprotein suspected to play a pivotal role in fundamental biological processes in cell proliferation and differentiation). Lane, Br. Med. Bull. 50:(3)582-599 (1994) also teaches the p53 gene involvement in various types of tumors. In addition, Lowe et al., Cell 74:957-967 (1993); see also Lowe et al., Science 266:807-810 (1994); Kastan et al., Cancer Res. 51:6304-6311 (1991); Fritsche et al., Oncogene 8:307-318 (1993) disclose that p53 activation is an important factor in mediating the cytotoxic effects of many cancer treatments, including chemotherapy and radiation, and that p53 is required to trigger apoptosis in response to chemotherapy.
Further elucidation of the role of both RB and p53 regulation has led to the mouse double-minute, or mdm2 oncogene. The human cDNA sequence (SEQ ID NO: 1) is disclosed in Volgelstein and Kinzler (U.S. Pat. No. 5,411,860) and the mouse cDNA sequence (SEQ ID NO: 12) can be found in GenBank, Accession No. U40145. Cahill-Snyder et al., Somatic Cell. Mol. Genet. 13:235-244 (1987) teach the identification of this oncogene because of its overexpression in a spontaneously transformed tumor cell line. Fakharzadeh et al., EMBO J. 10:1565-1569 (1991) disclose the ability of the mdm2 gene to augment tumorigenesis in NIH3T3 cells and Rat2 cells when overexpressed. More recent studies teach that mdm2 gene amplification and its subsequent overexpression occur frequently in a variety of tumors including soft tissue sarcomas, osteosarcomas, leukemias and gliomas Cordon-Cardo et al., Cancer Res. 54:794-799 (1994); Ladanyi et al., Cancer Res. 53:16-18 (1993); Leach et al., Cancer Res. 53:2231-2234; (1993); Oliner et al., Nature 358:80-83 (1992); Reifenberger et al., Cancer Res. 53:2736-2739 (1993); Sheikh et al. Cancer Res. 53:3226-3228 (1993); Matsumura et al., Oncology 53:308-312 (1996); Bueso-Ramos et al., Blood 82:2617-2623 (1993); Watanake et al., Blood 84:3158-3165 (1994).
Recently, investigators have sought to elucidate the mechanisms responsible for mdm2 putative oncogenicity and its interactions with tumor suppressors. Xiao et al., Nature 375:694-698 (1995) teach that the oncogenic activity of mdm2 is due, at least in part, to its ability to bind and inhibit p53 and RB transcriptional activation. Chen et al., Mol. Cell. Biol. 13:(7)4107-4114 (1993) have disclosed that p53 inactivation is due to the formation of a tight complex between the amino terminus of MDM2 and the amino terminal transactivation domain of p53. Chen et al., Mol. Cell. Biol. 16:2445-2452 (1996) has also disclosed that MDM2 inhibits G.sub.1 arrest and the apoptotic functions of p53. In addition, Wu et al., Genes Dev. 7:1126-1132 (1993) disclose that mdm2 is transcriptionally activated by p53, thus forming an autoregulatory negative feedback loop.
The MDM2 protein has also been shown to interact with other tumor suppressors and other molecules. Xiao et al. (supra) Martin et al., Nature 375:691-694 (1995) have recently disclosed the involvement of the same domain in the amino terminus of the MDM2 protein in the transcriptional activation of E2F1 DP1 further speculating a synergistic stimulation of the transcriptional activity of E2F1DP1 by relieving the negative control of RB on E2F1.
The significance of MDM2 in cell regulatory functions has recently been extended to other interactions. Marechal et al., Mol. Cell. Biol. 14:7417-7429 (1994) teach that the MDM2 protein binds to the ribosomal protein L5-5S RNA complex while Elenbaas et al., Mol. Med. 2:(4)439-451 (1996) teach MDM2 interaction with specific RNA structures.
From the available literature, it is clear that efforts should be directed to identify modulators and potentiators of tumor suppressor genes expression as a possible therapeutic approach to tumorigenesis. The identification of regulatory proteins acting on tumor suppressors could potentially lead to the development of therapeutic approaches to tumorigenesis by the activation of tumor suppressor functions. Thus, there is a need for the identification of tumor suppressor regulators and of methods to activate tumor suppressors in the context of chemotherapy. In this context, there is a need to elucidate the mechanism(s) involved in the development of resistance to chemotherapy in tumor cells. There is therefore, a need to develop better tools to carry out such investigations. Ideally, such tools should take the form of improved antisense oligonucleotides that inhibit mdm2. Kondo et al., Oncogene 10:(10)2001-2006 (1995) has disclosed that antisense oligonucleotide phosphodiesters directed against mdm2 increase the susceptibility of tumor cells to cisplatin-induced apoptosis. Kondo et al. have recently disclosed that mdm2 gene induced the expression of the multidrug resistance gene (mdr1) and that of its product P-glycoprotein (P-gp) conferring resistance to the apoptopic cell death induced by DNA-damage inducing drugs. Kondo et al., Br. J. Cancer 74:(8)1263-1268 (1996) teach the antisense inhibition of the mdm2 gene to inhibit expression of p-gp in mdm2 expressing glioblastoma cells further suggesting that the mdm2 gene may play an important role in the development of MDR phenotype in human tumors. Unfortunately the oligonucleotides disclosed arc phosphodiester oligonucleotides and thus not suitable as investigative tools for the purposes discussed herein, and as potential therapeutics for the treatment of ncoplastic diseases. Therefore, there remains a need for improved antisense oligonucleotides. Such improved antisense oligonucleotides should preferably also represent potential therapeutics for the treatment of neoplastic disease.